BRCA1 haploinsufficiency for replication stress suppression in primary cells

BRCA1—a breast and ovarian cancer suppressor gene—promotes genome integrity. To study the functionality of BRCA1 in the heterozygous state, we established a collection of primary human BRCA1+/+ and BRCA1mut/+ mammary epithelial cells and fibroblasts. Here we report that all BRCA1mut/+ cells exhibited multiple normal BRCA1 functions, including the support of homologous recombination- type double-strand break repair (HR-DSBR), checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. In contrast, the same cells were defective in stalled replication fork repair and/or suppression of fork collapse, that is, replication stress. These defects were rescued by reconstituting BRCA1mut/+ cells with wt BRCA1. In addition, we observed ‘conditional’ haploinsufficiency for HR-DSBR in BRCA1mut/+ cells in the face of replication stress. Given the importance of replication stress in epithelial cancer development and of an HR defect in breast cancer pathogenesis, both defects are candidate contributors to tumorigenesis in BRCA1-deficient mammary tissue

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism.

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases

IGH 3’RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sμ) and the 3’ regulatory region (3’RR) (Sμ-3’RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sμ-3’RRrec counts per sample: Sμ-3’RRrecHigh cases (mostly unmutated CLL) and Sμ-3’RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sμ-3’RRrec appeared to be ongoing in Sμ-3’RRrecHigh CLL cells and comparison of Sμ-3’RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sμ-3’RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sμ-3’RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sμ-3’RRrec in Sμ-3’RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sμ-3’RRrec, even in the absence of AID for the latter

Large deletions in immunoglobulin genes are associated with a sustained absence of DNA Polymerase η

International audienceSomatic hypermutation of immunoglobulin genes is a highly mutagenic process that is B cell-specific and occurs during antigen-driven responses leading to antigen specificity and antibody affinity maturation. Mutations at the Ig locus are initiated by Activation-Induced cytidine Deaminase and are equally distributed at G/C and A/T bases. This requires the establishment of error-prone repair pathways involving the activity of several low fidelity DNA polymerases. In the physiological context, the G/C base pair mutations involve multiple error-prone DNA polymerases, while the generation of mutations at A/T base pairs depends exclusively on the activity of DNA polymerase η. Using two large cohorts of individuals with xeroderma pigmentosum variant (XP-V), we report that the pattern of mutations at Ig genes becomes highly enriched with large deletions. This observation is more striking for patients older than 50 years. We propose that the absence of Pol η allows the recruitment of other DNA polymerases that profoundly affect the Ig genomic landscape

Proliferation and ovarian hormone signaling are impaired in normal breast tissues from women with <i>BRCA1</i> mutations: benefit of a progesterone receptor modulator treatment as a breast cancer preventive strategy in women with inherited <i>BRCA1</i> mutations

International audienceWomen with inherited BRCA1 mutations have an elevated risk (40-80%) for developing breast and ovarian cancers. Reproductive history has been reported to alter this risk, suggesting a relationship between ovarian hormone signaling and BRCA1-related tumor development. BRCA1 interactions with estrogen receptor (ER) and progesterone receptor (PR) signaling were previously described in human breast cancer cell lines and mouse models. However, few studies have examined the effect of ovarian hormone regulation in normal human breast tissues bearing a heterozygous BRCA1 mutation. This study compares the proliferation level (Ki67) and the expression of ER, PR, and of the PR target gene, fatty acid synthase (FASN), in histologically normal breast tissues from women with BRCA1 mutations (BRCA1+/mut, n=23) or without BRCA1 mutations (BRCA1+/+, n=28). BRCA1+/mut tissues showed an increased proliferation and impaired hormone receptor expression with a marked loss of the PR isoform, PR-B. Responses to estradiol and progesterone treatments in BRCA1+/mut and BRCA1+/+ breast tissues were studied in a mouse xenograft model, and showed that PR and FASN expression were deregulated in BRCA1+/mut breast tissues. Progesterone added to estradiol treatment increased the proliferation in a subset of BRCA1+/mut breast tissues. The PR inhibitor, ulipristal acetate (UPA), was able to reverse this aberrant progesterone-induced proliferation. This study suggests that a subset of women with BRCA1 mutations could be candidates for a UPA treatment as a preventive breast cancer strategy

<i>Bahd1</i>-knockout mice display decreased weight and fat mass and lower cholesterol, glucose and leptin levels.

<p><b>A-B.</b> Plasma levels of glucose (A) or of total cholesterol, HDL and LDL (B) in <i>Bahd1</i> wild-type (WT) and -heterozygous (HET) male (top) or female (bottom) mice, 10-week-old fed a chow diet (CD) or 30-week-old fed 14 weeks CD followed by 16 weeks HFHC (n = 8-10/group. <b>C.</b> Representative macroscopic images of <i>Bahd1</i>-WT and -knockout (KO) mice at 6 weeks and 9 months. <b>D.</b> Body length at 9 months. <b>E</b>. Body weight curve of <i>Bahd1-</i>WT and KO mice between the ages of 7 to 17 months (n = 5/group). <b>F.</b> Quantification of body fat mass and lean by QNMR analysis on 15 month-old animals (n = 4/group). <b>G.</b> Plasma levels of glucose, total cholesterol, HDL, insulin, leptin and adiponectin in 16-hours fasted 7-month-old WT and KO mice fed a normal diet (n = 4/group). <b>H.</b> Plasma levels of the same parameters in the same animals one year later (18 month-old). All data are expressed as the mean ± SE (* <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i>< 0.005).</p

Loss or overexpression of BAHD1 alters expression of genes involved in steroid metabolism.

<p><b>A.</b> Number of genes up- and down-regulated in <i>Bahd1</i>-KO placentas or MEFs relative to WT counterparts. <b>B.</b> Gene ontology enrichment analysis of transcripts differentially expressed in <i>Bahd1</i>-KO relative to WT placentas and MEFs, and in human HEK-BAHD1 cells relative to HEK-CT cells. The enrichment score represents the negative logarithm of the <i>p</i>-value evaluating the significance of gene ontology terms for differentially expressed RNAs. The top 10 annotation clusters are listed as derived from the DAVID bioinformatics tool. <b>C.</b> Transcripts levels in <i>Bahd1</i>^−/− placentas relative to <i>Bahd1</i>^+/+ littermates at E16.5 (n = 6, with 3 males and 3 females for each genotype) or E18.5 (n = 3 for each genotype) were quantified by RT-qPCR. Values are normalized by <i>Gapdh</i>. <i>Ywhaz</i> is used as negative control. The differential expression in <i>Bahd1</i>^−/− placentas is shown (relative to that in <i>Bahd1</i>^+/+ = 1). <b>D.</b> Relative transcripts levels for several genes in <i>Bahd1</i>^−/− relative to <i>Bahd1</i>^+/+ placentas at E18.5 (n = 3 for each genotype). <b>E.</b> Relative transcripts levels for imprinted genes in <i>Bahd1</i>^−/− relative to <i>Bahd1</i>^+/+ placentas at E16.5 (n = 4 for each genotype). Values are normalized to <i>Hprt</i>, <i>ActB and Tuba1a</i>. <b>F</b>. Venn diagram depicting shared genes that are up-regulated in <i>Bahd1</i>-KO E18.5 placentas and MEFs and down-regulated in human HEK-BAHD1 cells. <b>G</b>. Relative transcripts levels for lipid metabolism genes in HPT-BAHD1 cells relative to control HPT-CT. <i>BAHD1</i> expression was induced with tetracycline for 30h. Data are expressed as mean ± SD (ns, non-significant; * <i>P</i> < 0.05; ** <i>P</i> < 0.005; *** <i>P</i> < 0.001).</p

BAHD1 increases MIER1 nuclear translocation and recruitment to Xi.

<p><b>A.</b> Cytoplasmic, nuclear soluble or chromatin extracts from HPT-control or HPT-BAHD1 cells induced 30 h with doxycyline were analyzed by immunoblotting with BAHD1, MIER1, histone H3 or Tubulin-α antibodies. H3 and Tubulin were used as loading controls. <b>B.</b> Colocalization of Protein-C tagged-BAHD1 and MIER1 on heterochromatic Xi (pointed with arrows on the merged image) was examined by IF with Protein-C and MIER1 antibodies. Bar, 5 μm. <b>C.</b> Localization of BAHD1 and MIER1 in HPT-BAHD1 cells induced or not for 30 h with doxycycline. Enrichment on Xi was determined by IF with BAHD1 (top panels) or MIER1 (bottom panels) antibodies combined with Xist RNA FISH (red). DNA was stained with DAPI (blue in the merged image). Bars: 10 μm.</p

Depletion or overexpression of BAHD1 in HEK293 cells induce epigenetic changes at <i>ESR1</i>.

<p><b>A. BAHD1 and MIER1/3 repress <i>ESR1</i> and <i>PGR</i></b>. HEK293-FT cells were transfected for 72 h with control or BAHD1, MIER1 or MIER3 siRNA. The levels of <i>BAHD1</i>, <i>MIER1</i>, <i>MIER3</i>, <i>ESR1</i>, <i>PGR</i> and <i>AR</i> transcripts were quantified by RT-qPCR. Data are expressed as mean ± SD (* <i>P</i><0.05; ** <i>P</i><0.005). <b>B.</b> Schematic representation of the proximal region of <i>ESR1</i> in chr6 of the human genome. <b>C.</b> BAHD1 binds the proximal region of <i>ESR1</i>. Amounts of DNA precipitated with BAHD1-TAP E1 eluates (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005898#pgen.1005898.g004" target="_blank">Fig 4</a>) or with control TAP or inputs were quantified using qPCR with the primer sets indicated in B. The amount of DNA purified with BAHD1 was normalized to the amount precipitated in the control TAP, and to the <i>GAPDH</i> locus. Data are averages ± SD of qPCR triplicates, and representative of 2 independent TAP experiments. <b>D.</b> BAHD1 depletion alters the patterns of H3K9 acetylation and methylation at the <i>ESR1</i> locus. HEK293-FT cells were transfected with control or BAHD1 siRNA and enrichment of H3K9ac, H3K9me2 and H3K9me3 relative to IgG control at <i>ESR1</i> and <i>GAPDH</i> regions were estimated by ChIP-qPCR in BAHD1-depleted and control cells. The y-axis shows the relative fold change of ChIP enrichment in cells with BAHD1 siRNA over cells with control siRNA in Log2 ratios. Data are averages ± SD of two ChIP per antibody and representative of three biological replicates (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005898#pgen.1005898.s007" target="_blank">S6 Fig</a>). <b>E.</b> Overexpression of BAHD1 induces widespread DNA methylation at the <i>ESR1</i> locus. Bisulfite-modified genomic DNA of control HEK-CT cells and isogenic HEK-BAHD1 cells overexpressing BAHD1 were sequenced and analyzed for their DNA methylation status, as described in Libertini et al. (2015) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005898#pgen.1005898.ref014" target="_blank">14</a>]. 300bp-regions with reproducible gain of methylation in HEK-BAHD1 compared to HEK-CT DNA in two BS-seq replicates (i.e hypermethylated BAHD1-DMRs) were binned into 0.5Mb windows highlighting clusters of hyper-DMRs (shown as red bars). Contiguous clusters define BAHD1-associated domains (“Hyper-BADs”, shown as black boxes). BAHD1-DMRs are represented by black vertical lines in the track “hyper-DMRs”. Three hypermethylated BADs of chr6 are shown on the top, with the position of <i>ESR1</i> indicated by an arrow. A 1 Mb region encompassing the <i>Esr1</i> locus is magnified below, showing the high density of hypermethylated BAHD1-DMRs on the whole locus. CpG islands are indicated in green. The position of transcripts is shown below.</p